Poject Summary Diet strongly influences the risk of developing a metabolic disease, a digestive disorder, and/or cancer, but surprisingly little is known about the underlying mechanisms. Organismal dietary status is processed at the cellular level by mechanistic Target of Rapamycin, or mTOR, a major regulator of eukaryotic cell growth, proliferation, and survival in response to nutrients, growth factors, energy levels, and stress. Indeed, dysregulated mTOR activity is frequently observed in disease states with strong dietary risk factors such as Type II Diabetes and colon cancer. Adult stem cells, vital for maintaining tissue homeostasis throughout life, are also sensitive to diet and in appropriate stem cell activity has been linked to cancer and digestive disorders such as intestinal failure and short gut syndrome. mTOR serves as a link between diet and stem cell activity in lower organisms, such as Drosophila, but no studies have been published investigating the role of this highly therapeutically-targeted signaling pathway in an adult mammalian stem cell population. The intestine is a diet- responsive, highly-proliferative, stem-cell based tissue with an organized epithelium of crypts (small intestine and colon) and villi (small intestine). Intestinal stem cells (ISCs) are localized at the crypt base and differentiated progeny move up from the crypts to the villi making this highly ordered model ideal for studies of adult stem cell regulation. We propose that mTOR, through two downstream signaling complexes with roles in translational regulation (mTORC1) and cytoskeletal dynamics (mTORC2), regulates ISC proliferation, long- term self-renewal, metabolic function, and progenitor differentiation potential. These various ISC characteristics will be examined in mTOR and mTOR signaling complex, mTORC1 (Raptor) and mTORC2 (Rictor), constitutive and inducible intestine-specific mutant mice. These studies will allow the assignment of mTOR mutant phenotypes as either mTORC1 or mTORC2-mediated and could identify intestinal roles for mTOR independent of the two known signaling complexes-a finding that would revolutionize the mTOR signaling field. These in vivo studies will be complemented by in vitro intestinal organoid culture assays that together will provide a detailed, highly-controlled analysis of the roles of mTOR via mTORC1 or mTORC2 in regulating ISC proliferation, self-renewal, and production of differentiated progeny. These studies will provide targeted knowledge on the role of diet in disease initiation and/or progression via effects on stem cell activity, insight that is sorely needed in light of the strain of metabolic disease, digestive disorders, and cancer on human health and the economy.